Neuroinflammation, autoinflammation, splenomegaly and anemia caused by bi-allelic mutations in IRAK4

Abstract

We describe a novel, severe autoinflammatory syndrome characterized by neuroinflammation, systemic autoinflammation, splenomegaly, and anemia (NASA) caused by bi-allelic mutations in IRAK4. IRAK-4 is a serine/threonine kinase with a pivotal role in innate immune signaling from toll-like receptors and production of pro-inflammatory cytokines. In humans, bi-allelic mutations in IRAK4 result in IRAK-4 deficiency and increased susceptibility to pyogenic bacterial infections, but autoinflammation has never been described. We describe 5 affected patients from 2 unrelated families with compound heterozygous mutations in IRAK4 (c.C877T (p.Q293*)/c.G958T (p.D320Y); and c.A86C (p.Q29P)/c.161 + 1G>A) resulting in severe systemic autoinflammation, massive splenomegaly and severe transfusion dependent anemia and, in 3/5 cases, severe neuroinflammation and seizures. IRAK-4 protein expression was reduced in peripheral blood mononuclear cells (PBMC) in affected patients. Immunological analysis demonstrated elevated serum tumor necrosis factor (TNF), interleukin (IL) 1 beta (IL-1β), IL-6, IL-8, interferon α2a (IFN-α2a), and interferon β (IFN-β); and elevated cerebrospinal fluid (CSF) IL-6 without elevation of CSF IFN-α despite perturbed interferon gene signature. Mutations were located within the death domain (DD; p.Q29P and splice site mutation c.161 + 1G>A) and kinase domain (p.Q293*/p.D320Y) of IRAK-4. Structure-based modeling of the DD mutation p.Q29P showed alteration in the alignment of a loop within the DD with loss of contact distance and hydrogen bond interactions with IRAK-1/2 within the myddosome complex. The kinase domain mutation p.D320Y was predicted to stabilize interactions within the kinase active site. While precise mechanisms of autoinflammation in NASA remain uncertain, we speculate that loss of negative regulation of IRAK-4 and IRAK-1; dysregulation of myddosome assembly and disassembly; or kinase active site instability may drive dysregulated IL-6 and TNF production. Blockade of IL-6 resulted in immediate and complete amelioration of systemic autoinflammation and anemia in all 5 patients treated; however, neuroinflammation has, so far proven recalcitrant to IL-6 blockade and the janus kinase (JAK) inhibitor baricitinib, likely due to lack of central nervous system penetration of both drugs. We therefore highlight that bi-allelic mutation in IRAK4 may be associated with a severe and complex autoinflammatory and neuroinflammatory phenotype that we have called NASA (neuroinflammation, autoinflammation, splenomegaly and anemia), in addition to immunodeficiency in humans.

Keywords: IRAK-4; NASA; anemia; autoinflammation; neuroinflammation; splenomegaly; toll-like receptor.

Figure 1

Pedigrees, genetic analysis, neurological features and shedding assay results in families A and B. (A, B) Pedigrees of family A and family B, respectively, with black shapes showing affected children, white shapes showing unaffected parents and children and arrows indicating the proband of each family. Genetic testing revealed that the affected children in family A were compound heterozygous for IRAK4 gene mutations c.C877T (p.Q293*) and c.G958T (p.D320Y) and in family B were compound heterozygous for IRAK4 gene mutations c.A86C (p.Q29P) and c.161 + 1G>A. Parents in both families were found to be heterozygotes. (C) Sanger sequencing electropherograms for family A showing the c.C877T (p.Q293*) variant inherited from the father (A-I-1), and c.G958T (p.D320Y) variant inherited from the mother (A-I-2). (D) Sanger sequencing electropherogram for family B showing c.A86C (p.Q29P) variant inherited from the father (B-I-1), and c.161 + 1G>A variant inherited from the mother (B-I-2). (E) Brain imaging. Left column (A-II-2): Axial CT scan showing bitemporal subcortical calcification (arrows). Post-contrast coronal FLAIR (FLAIR+C) and axial T1-weighted (T1+C) MRI sequences, showing asymmetrical swelling, signal abnormality and cortical-subcortical contrast enhancement in temporal lobes, insular and basal frontal regions. Right column (B-II-2): CT scan showing patchy subcortical calcification in the temporal lobes bilaterally (arrows). Coronal MRI FLAIR demonstrates asymmetrical signal abnormality involving the insular regions and temporal lobes. Post-contrast T1-weighted (T1+C) MRI performed after 3 months shows persisting cortical and subcortical contrast enhancement with signal abnormality in the temporal lobes and the frontal basal regions. (F) Brain biopsy of B-II-2 showing disruption of the cortical architecture with interspersed reactive astrocytes in keeping with gliosis (I); blood vessels with reactive endothelium and scattered mononuclear chronic inflammation (II); scattered CD3 positive T lymphocytes (III); diffuse microglial upregulation with interspersed macrophages (IV).

Figure 2

Shedding assay results. CD62L shedding was assessed by flow cytometry results for CD11b positive granulocytes from affected patients A-II-1, A-II-2, and B-II-3 and healthy adult controls following Toll-like receptor (TLR) agonists LPS (TL4), MALP-2 (TLR2/6), Flagellin (TLR5), PAM3CSK5 (TLR1/2) and R-848 (TLR7/8) stimulation. Phosphate buffered saline (PBS) and phorbolmyristyl acetate (PMA) were used as negative and positive controls, respectively. The percentage of gated granulocytes that have shed CD62L is indicated in the top left-hand quadrant.

Figure 3

Location and structural modeling of IRAK-4 mutations and impact on protein expression. (A) Schematic representation of the 460 amino acid IRAK-4 protein showing the N-terminal death domain (DD; green), the C-terminal kinase domain (KD; blue) and within the KD domain the hinge region (H; grey). The D320Y and Q29P mutations identified in family A and family B, respectively are indicated by black arrows. Multiple sequence alignment of IRAK-4 amino acid sequences surrounding Q29 within the DD and D320 within KD of IRAK-4 from different species (yellow). The location of α-helices 1 and 2 and the activation segment (pink) are also indicated. (B) Superposition of mutant Pro29 and wild type Gln29 between α-helices 1 and 2 (α1 and α2) in the structure of the death domain of IRAK-4. Adjacent residues Asp27 and Pro28 are also shown. (C) Analysis of the interface between p.Q29P IRAK-4 and IRAK-2, compared to wild type IRAK-4, showing increased distance with loss of hydrogen bond and hydrophobic interactions. (D) Schematic representation of myddosome assembly of DD of MyD88 (M¹-M⁶), IRAK-4 (I4¹-I4⁴), and IRAK-2 (I2¹-I2⁴) and the predicted impact of the p.Q29P on the interaction between IRAK-4 (I4mut³) and IRAK-2 (I2³) and interacting interface II of I-III. (E) Superposition of mutant Tyr320 and wild type Asp320 located in the loop region between β-sheets 6 and 7 (β6 and β7) in the kinase domain structure of IRAK-4. Analysis of interaction of atoms of Asp320 and mutant Tyr320 residues were examined within a radius of 4.5 Å using a protein contacts panel, showing hydrophobic interaction with Met265. (F) Flow cytometry analysis of intracellular IRAK-4 expression in patient derived PBMCs. Healthy controls (black circles) and wild type patient (B-II-4; lower circle), heterozygous parents (grey inverted triangles) and compound heterozygotes from family A (grey circles) and family B (grey squares) are shown. (G) Representative flow cytometry histograms of IRAK-4 expression (median fluorescence intensity APC, allophycocyanin) in lymphocytes and monocytes from an affected patient (purple) versus a healthy control (dashed) are shown compared to unstained cells (grey). Statistical significance is indicated by p-values (*0.01 and** 0.001 in lymphocytes and *0.03 and **0.007 in monocytes), with non-significance indicated by ns.

Figure 4

Functional evaluation of IRAK-4 signaling. (A) Pro-inflammatory cytokine quantification in sera using Meso Scale Discovery. Healthy controls (black circles), heterozygote parents (dark grey inverted triangles) and compound heterozygotes from Family A (light grey squares) and Family B (light grey diamonds), are shown. (B) NF-κB signaling assessed via flow cytometry analysis of phosphorylated-p65 (P-p65) stimulated with ligands as indicated on x-axis, fold-change median fluorescence (MFI) from baseline at 20 minutes shown. (C) Analysis of interleukin-6 (IL-6) and tumor necrosis factor (TNF) production by peripheral blood mononuclear cells (PBMC) from Family A in response to lipopolysaccharide (LPS) stimulation. (D) STAT1 and (E) STAT3 signaling assessed via flow cytometry analysis of phosphorylated-STAT1 (p-STAT1) and STAT3 (p-STAT3) expression following stimulation with interferon α2b (IFN-α2b) at time points indicated on x axis. Horizontal lines at mean and standard error (SE) of the mean shown. Significance indicated by: *p < 0.05; ** p < 0.01; and ****p < 0.0001 and ns, not significant. TNF, tumor necrosis factor; IL, interleukin; IP-10, interferon-γ induced protein-10; MCP-1, monocyte chemoattractant protein-1; IFN, interferon; LPS, lipopolysaccharide; p-STAT1/3, phosphorylated signal transducer and activator of transcription 1/3; MFI, median fluorescence intensity.